Method for measuring the amount of constituent contained in a specific lipoprotein

ABSTRACT

There is provided a method and kit for measuring the amount of an objective constituent contained in a specific lipoprotein in a biological sample such as serum and plasma, specifically for measuring the amount of cholesterol contained in high density lipoprotein, which can be applicable to clinical tests.

This application is a Continuation of prior application Ser. No.09/693,891, filed Oct. 23, 2000, now abandoned, which is a Divisional ofprior application Ser. No. 08/684,298, filed Jul. 19, 1996, now U.S.Pat. No. 6,162,607, which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a method for measuring the amount of anobjective constituent for measurement contained in a specificlipoprotein in a biological sample such as serum and plasma. Moreparticularly, the invention provides a method for measuring the amountof cholesterol contained in high density lipoprotein (hereinafterreferred to as HDL). The method of the present invention can be appliedadvantageously to automatically quantitative measurement of HDLcholesterol which has widely been used in the field of clinical tests.

As to methods for measuring the amount of the objective constituent formeasurement contained in the specific lipoprotein such as cholesterolcontained in HDL, there have been known various methods includingultracentrifugal separation method, electrophoresis method,precipitation method and the like. In the field of clinical tests, theprecipitation method has usually and widely been carried out, for thereason that this method can be carried out simply as compared with theultracentrifugal separation method and the electrophoresis method.

However, a problem with conducting the precipitation method is that thismethod cannot be carried out only by use of a conventional automaticanalyzer, for the reason that this method comprises two steps; (1) astep of pretreatment operation, which comprises, a) mixing a serum witha precipitating agent and forming precipitates of the lipoproteins otherthan the specific lipoprotein, b) separating the precipitates bycentrifugation, and c) collecting thus formed supernatant; and (2) astep of measuring the amount of the objective constituent contained inthe specific lipoprotein in the supernatant.

In order to solve the problem, there have been developed some methods;for example Japanese Patent Kokai (Laid-open) No. Hei 6-242110 (1994)discloses a method in that, lipoproteins other than the specificlipoprotein are agglutinated by use of a precipitating agent and/or anantibody reactive to the lipoproteins other than the specificlipoprotein, then the specific constituent in the specific lipoproteinis subjected to reaction with a reagent for quantitative measurementthereof, and thereafter, at the same time, or after the reaction isstopped, the agglutinated lipoprotein is dissolved to give a homogeneoussolution, and then the optical absorbance of said solution is measured.

This method, however, requires 3 or 4 kinds of reagents and thus can beapplicable only to such remarkably limited automatic analyzers whereinmeasurement can be conducted with the use of 3 or 4 kinds of reagents,while this method cannot be applied to such automatic analyzers whereinonly up to 2 kinds of reagents can be used as conventionally used inclinical tests. Further, this method has the defect of lowerreproducibility of measurement because of using 3 or 4 kinds ofreagents.

In consideration of the above circumstances, the problem to be solved bythe present invention is to provide a method capable of measuringdirectly the amount of the objective constituents for measurementcontained in the specific lipoprotein in a biological sample by using anautomatic analyzer, without subjected to any pretreatment operation toseparate the specific lipoprotein from other lipoproteins, which hasnecessarily been carried out in conducting the precipitation methodwidely used in conventional clinical tests.

SUMMARY OF THE INVENTION

The present invention relates to a method for measuring the amount of anobjective constituent for measurement contained in a specificlipoprotein among lipoproteins contained in a biological sample, whichcomprises mixing a biological sample with an antibody reactive tolipoprotein(s) other than said specific lipoprotein and thereaftermeasuring the absorbance (OD₁) of the reaction mixture, and then mixingsaid reaction mixture with a reagent for measurement of the objectiveconstituent for measurement contained in the lipoprotein and thereaftermeasuring again the absorbance (OD₂) of the latter reaction mixture andcalculating the amount of the objective constituent for the measurementcontained in the specific lipoprotein on the basis of the differencebetween OD₁ and OD₂.

Furthermore, the present invention relates to a kit for measurement ofan objective constituent for measurement contained in the specificlipoprotein, which comprises (i) a reagent composition containing anantibody reactive to lipoprotein(s) other than the specific lipoproteinand a buffering agent and (ii) a reagent composition containing areagent for measurement of the objective constituent for measurementcontained in a lipoprotein and a buffering agent.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have made an extensive research work for finding amethod for measuring directly the amount of an objective constituentcontained in a specific lipoprotein by use of an automatic analyzerwithout carrying out any pretreatment operation to remove thelipoproteins other than the specific lipoprotein.

As a result, the inventors have found that measurement of constituent(s)in a specific lipoprotein without previously removing lipoproteins otherthan the specific lipoprotein is possible by mixing a biological samplewith a first reagent solution comprising an antibody reactive tolipoprotein(s) other than the specific lipoprotein to allow a reactionto take place, and thereafter measuring an absorbance (OD₁) of thereaction mixture, and then mixing the reaction mixture with a secondreagent solution comprising a reagent for measurement of the constituentin the lipoprotein to allow a reaction to take place, and thereaftermeasuring an absorbance (OD₂) of the latter reaction mixture and usingthus measured absorbance OD₁ and OD₂, and on the basis of thisdiscovery, the present invention has been established.

The lipoproteins are classified, according to their specific gravities,into chylomicrons, very low density lipoprotein (VLDL), low densitylipoprotein (LDL) and HDL and the like.

In the present invention, any one of these lipoproteins can be selectedas the specific lipoprotein to be the object of the measurement. And formeasurement of the amount of any specific constituent contained in thusselected specific lipoprotein, first a biological sample is mixed withan antibody reactive to lipoprotein(s) other than the specificlipoprotein to allow a reaction to take place, then the opticalabsorbance (OD₁) of the thus obtained reaction mixture is measured, andnext said reaction mixture is mixed with a reagent for measuring thespecific constituent for measurement to allow a reaction to take place,then the optical absorbance (OD₂) of the latter reaction mixture ismeasured, and the amount of the objective constituents for measurementcontained in the specific lipoprotein is determined based on themeasured values of the optical absorbances (OD₂) and (OD₁), withoutconducting any operation for removing lipoproteins other than thespecific lipoprotein.

In the present invention, the objective constituents for measurementcontained in the specific lipoprotein may include but are not limited tocholesterol, triglycerides, phospholipids and the like.

The antibody reactive to lipoprotein(s) other than the specificlipoprotein in the present invention may include any ones such as havingan effect of preventing the objective constituents for measurementcontained in lipoproteins other than the specific lipoprotein fromparticipating in the reaction with a reagent for measuring the amount ofthe objective constituents contained in the lipoprotein, and theantibody may be monoclonal antibody or polyclonal antibody, regardlessof its origin, so far as it has the effect mentioned above. Taking thedegree of the preventing effect into consideration, however, apolyclonal antibody is preferable. When a monoclonal antibody is used,it is preferable to use 2 or 3 kinds or more, preferably 5 kinds or moreof monoclonal antibodies which recognize different antigenicdeterminants from each other. Examples of the preferable antibody in thepresent invention include an anti-apolipoprotein antibody such asanti-apolipoprotein A, anti-apolipoprotein B, anti-apolipoprotein C andanti-apolipoprotein E, an anti-lipoprotein antibody such asanti-αlipoprotein antibody and anti-βlipoprotein antibody.

The most preferable antibody is selected from those mentioned above, inaccordance with the objective lipoprotein(s) for measurement.

For instance, in case of measuring the objective constituents of HDL,the antibody to be used for the purpose of preventing the objectiveconstituent contained in lipoproteins other than HDL from participatingin the measuring reaction, may include anti-apolipoprotein B antibody,anti-apolipoprotein C antibody, anti-apolipoprotein E antibody,anti-βlipoprotein antibody and the like.

These antibodies may be used singly or in combination by mixing suitablywith any one or more of these, so far as said antibody has the desiredpreventing effect mentioned above. Furthermore, these antibodies may bethose of enzymatically or chemically decomposed or modified types, suchas F(ab′)2, enzyme-antibody conjugate and hapten-antibody conjugate, andthe like.

The antibody of the present invention is one which reacts withlipoprotein(s) other than the specific lipoprotein so as to prevent theobjective constituents for measurement contained in lipoproteins otherthan the specific lipoprotein from participating in the reaction.

Among the antibodies, such ones are preferable that agglutination causedby the reaction of the antibody with lipoprotein(s) other than thespecific lipoprotein is not so high as hindering the desiredmeasurement.

The concentration of the antibody reactive to lipoprotein(s) other thanthe specific lipoprotein, which is to be used, is not specificallyrestricted, so far as being higher than that capable of preventing theobjective constituent for measurement contained in the lipoproteinsother than the specific lipoprotein from reacting with the reagent formeasuring the objective constituent, and in general, the antibody isadded to the first reagent solution so as to keep the concentrationthereof in a mixture of the first and the second reagent solutions andthe biological sample to be measured in the range of 0.001 to 10mgAb/ml, preferably 0.01 to 1 mgAb/ml.

It is preferable to incorporate a buffering agent in the reagentsolution comprising an antibody.

Any buffering agent can be used, so far as having buffering ability tokeep pH 5.0 to 11.0 and not inhibiting the measuring reaction of theobjective constituents for measurement, andtris(hydroxymethyl)-aminomethane, Good's buffering agent, phosphates,berates and the like can be exemplified. For instance, the bufferingagent to be used for measuring the amount of cholesterol in HDL isexemplified by those having the buffering ability to keep pH 5.7 to 9.1such as glycine derivatives including N-(2-acetamide)-2-aminoeth-anesulfonic acid and N-(2-acetamide)-imino-2-acetic acid, hydroxyalkylaminederivatives, more particularly, 2-hydroxyethylamine derivativesincluding N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid,bis(2-hydroxyethyl) imino-tris(hydroxymethyl)methane,3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid and the like,tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)amine derivativesincluding 3-[N-tris(hydroxymethyl)methylamine]-2-hydroxypropanesul fonicacid, N-tris(hydroxymethyl)-2-aminoethanesulfonic acid and the like, andthe hydroxyalkylamine derivatives are, among of them, particularlypreferable in view of measuring accuracy. Specifically, in case of usingfor measuring the amount of cholesterol in HDL, a hydroxyalkylaminederivative is preferable.

The concentration of the buffering agent to be used is generallyselected from a range of 10 mM to 1M, preferably 20 to 500 mM.

As the reagent for measuring the amount of the objective constituent,any one of known ones which have been used for measuring the amount ofthe objective constituent contained in the lipoprotein can be usedwithout any specific limitation. Furthermore, at present, variousmeasuring methods applying enzymes (i.e., for enzymatic methods) havewidely been used in this field, and reagents used for such enzymaticmethods are advantageously used in the present invention because oftheir easy availability.

For instance, as the reagent for measuring the amount of cholesterolcontained in the lipoprotein, there can be exemplified by those whichhave been used in known measuring methods, such as reagents foroxidizable-colorimetric method which comprises cholesterol oxidase(COD), cholesterol esterase (CHE), peroxidase (POD), oxidizable colorreagents, etc., and reagents for ultraviolet (UV) spectrometric methodwhich comprises CHE, cholesterol dehydrogenase (CHD), nicotinamideadenine dinucleotide (NAD).

As the reagents used for measuring the amount of triglycerides in thelipoprotein, there can be exemplified by those which have been used inknown measuring methods such as glycerokinase(GK)-glycerol-3-phosphateoxidase method, glyceroldehydrogenase method and GK-glycerol-3-phosphatedehydrogenase method.

As the reagents used for measuring the amount of phospholipid in thelipoprotein, there can be exemplified by those which have been used inknown measuring methods such as an organic solvent extraction method anda method using phospholipase and cholinoxidase.

Each one of these reagents for measuring the amount of the objectiveconstituents is generally comprised in the reagent solution containingno antibody (the 2nd solution), while some of which, for example POD, anoxidizable color reagent and NAD may be comprised in the reagentsolution containing the antibody (the 1st solution). The concentrationof each one of these reagents to be comprised in the reagent solutionscan be suitably selected from such a range as having been used in themeasurement in this field.

The reagent solution containing no antibody (the 2nd solution) used inthe present invention preferably further comprises a surfactant. Thesurfactant has the effect of promoting the measurement reaction of theobjective constituent contained in the specific lipoprotein,particularly cholesterol, so as to make it possible to shorten thereaction time. The surfactant to be added for this purpose can be,without any limitation, any of nonionic surfactants, amphotericsurfactants, cation surfactants and anion surfactants, so far as they donot hinder the measurement reaction of the objective constituentcontained in the specific lipoprotein. The surfactant includes nonionicsurfactants such as polyoxyethylene cetyl ether, polyoxyethylene oleylether, polyoxyethylene lauryl ether, polyoxyethylene alkylphenyl etherincluding polyoxyethylene isooctylphenyl ether and polyoxyethylenenonylphenyl ether, and polyethylene glycol monolaurate, and amphotericsurfactants such as stearyl betaine and2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine, and anionsurfactants such as choleic acid, deoxy cholic acid and sodiumpolyoxyethylene alkylphenol ether sulfate. Among those surfactants,nonionic ones having HLB value of 12 to 17 are preferable in view of theaccuracy of the measurement. As the nonionic surfactants used inmeasurement of cholesterol in HDL, ones having HLB value of 12 to 17 areparticularly preferable. Those surfactants can be used alone or insuitable combination.

The concentration of the surfactant to be used is not critical, and ingeneral, it is added to the 2nd solution in such an amount that thefinal concentration in the mixture of the 1st and the 2nd solutions andthe biological sample to be measured is 0.001 to 10 w/v %, preferably0.01 to 1 w/v %.

It is to be noted that when the surfactant is added to the firstsolution, measurement error caused by the objective constituentcontained in lipoproteins other than the specific lipoprotein may beobserved.

An agglutination promoter such as polyethylene glycol and polyvinylalcohol which has generally been used in measurement method usingimmunoagglutination reaction, is not required to be present in the 1stor the 2nd solution used in the measurement method of the presentinvention.

This does not mean, however, that addition of the agglutination promotershould completely be excluded, but the promoter may be incorporated inthe 1st and/or the 2nd solution, so far as the amount is such one as nothindering the desired measurement.

The measurement of the present invention is conducted, for instance, asmentioned below.

A biological sample such as serum and/or plasma is mixed with a firstsolution comprising an antibody reactive to lipoprotein(s) other thanthe specific lipoprotein and a buffering agent to allow a reaction totake place at 2 to 40° for 3 to 30 minutes, and thereafter an absorbance(OD₁) of the reaction mixture is measured. Then, the reaction mixture ismixed with the 2nd solution comprising a reagent for measuring theobjective constituent, a buffering agent and if necessary a surfactant,to allow a reaction to take place at 2 to 40° for 3 to 30 minutes, andthereafter an absorbance (OD₂) of the latter reaction mixture ismeasured.

The value of OD₁ is subtracted from OD₂ to give OD₃, and the obtainedOD₃ is applied to a calibration curve showing the relationship betweenthe concentration of the objective constituent for measurement and OD₃,which is previously prepared by conducting the same procedure as abovewith the use of the same reagents as above on standard solutionscontaining known amounts of the objective constituent for measurement inthe specific lipoprotein, whereby the amount of the objectiveconstituent for measurement in the biological sample is obtained. Whenthe volume of the solution to which measurement of OD₁ is conducted isdifferent from that to which the measurement of OD₂ is conducted, theOD₁ value is corrected in accordance with the difference before beingsubtracted from OD₂.

The kit of the present invention for measuring the amount of anobjective constituent in a specific lipoprotein comprises (i) a reagentcomposition (the first reagent composition) comprising an antibodyreactive to lipoprotein(s) other than the specific lipoprotein and abuffering agent, and (ii) a reagent composition (the second reagentcomposition) comprising the reagent for measuring the amount ofobjective constituent in the specific lipoprotein, a buffering agent andif necessary a surfactant, and the preferable embodiments of thosecomponents are as mentioned before.

A part of the components usually contained in the second reagentcomposition may be incorporated in the first reagent composition, so faras no hindering effect to the measurement is observed. For instance,when cholesterol in the specific lipoprotein is measured by an enzymaticmethod with the use of cholesterol oxidase (COD), cholesterol esterase(CHE), peroxidase (POD), a reagent developing color by coupling with4-aminoantipyrine by the act of 4-aminoantipyrine and an oxidizing agent(hereinafter abbreviated as coloring reagent) as the reagent formeasurement of cholesterol, it is essential to incorporate an antibodyagainst lipoprotein(s) other than the specific lipoprotein and abuffering agent in the first reagent composition and to incorporate CODand CHE in the second reagent composition, and the second reagentcomposition preferably contains also a surfactant, but other componentsmay be incorporated in the first or second reagent composition.

From view point of stability of the reagent compositions, use ispreferably made of a combination of the first reagent compositionincorporated with an antibody against lipoprotein(s) other than thespecific lipoprotein, a buffering agent and 4-aminoantipyrine and thesecond reagent composition incorporated with COD, CHE, POD, a coloringreagent, a buffering agent and a surfactant.

In the following, the present invention is further explained in detailswith citation of Examples and Reference examples, but the presentinvention is not limited to those examples in any way.

Abbreviations used in the examples mean as follow.

-   BES: N,N-bis(2-hydroxyethyl)-2-aminoethansulfonic acid-   COD: cholesterol oxidase CHE: cholesterol esterase-   POD: peroxidase-   DAOS: N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyanaline-   Triton X-100 (Trade Name; manufactured and sold by Rohm & Haas):    polyoxyethylene alkylphenyl ether (HLB: 13.5)-   ACES: N-(2-acetamido)-2-aminoethanesulfonic acid-   Emalex NPL-30 (Trade Name; manufactured and sold by Nihon-   Emulsion Co.): polyoxyethylene nonylphenyl ether (HLB: 17)-   Triton X-405 (Trade Name; manufactured and sold by Rohm & Haas):    polyoxyethylene alkylphenyl ether (HLB: 17.9)

EXAMPLE 1

Amount of cholesterol comprised in HDL in serum was measured by themethod of the present invention with the use of an automatic analyzerHitachi 7150 (Trade Name; manufactured and sold by Hitachi Ltd.).

[Samples]

10 samples of fresh human serum.

[Reagents]

1st reagent (R-1): Antiserum to βlipoprotein (12 mgAb/ml, 1% w/vmanufactured and sold by Wako Pure Chemical Industries, Ltd.)4-Aminoantipyrine 1 mM BES-NaOH buffer solution (pH 7.0) 100 mM 2ndReagent (R-2): COD 3 Units /ml CHE 3 Units /ml POD 1 Unit / ml DAOS 1 mMTriton X-100 0.05% w/v BES-NaOH buffer solution (pH 7.0) 100 mM[Measuring Parameters (Measuring Conditions)]

Measurement method: 2 Point end method [24]–[50] Sample volume: 4 μl R-1volume: 270 μl R-2 volume: 90 μl Measurement wave length: 700/600 nmMeasurement temperature: 37° C.[Results]

The measurement result is shown in the table 1.

EXAMPLE 2

Amount of cholesterol contained in HDL in serum was measured by themethod of the present invention with the use of an automatic analyzerHitachi 7150 (Trade Name; manufactured and sold by Hitachi Ltd.).

[Samples]

Same as Example 1.

[Reagents]

1st reagent (R-1): Antiserum to βlipoprotein (12 mgAb/ml, 1% w/vmanufactured and sold by Wako Pure Chemical Industries Ltd.)4-Aminoantipyrine 1 mM ACES-NaOH buffer solution (pH 7.0) 100 mM 2ndreagent (R-2): COD 3 Unit /ml CHE 3 Unit /ml POD 1 Unit /ml DAOS 1 mMTriton X-100 0.05% w/v ACES-NaOH buffer solution (pH 7.0) 100 mM[Measuring Parameters (Measuring Conditions)]

Same as Example 1.

[Results]

The measurement results is also shown in the table 1.

Reference Example 1

On the serum samples used in Example 1, cholesterol contained in HDL wasmeasured by known phosphorus-tungstic acid/magnesium salt precipitationmethod with the use of HDL-Cholesterol E-Test Wako (Trade Name;manufactured and sold by Wako Pure Chemical Industries Ltd.).

The measurement procedure was as the standard procedure explained in thebrochure of said kit.

[Results ]

The measurement result is also shown in the table 1.

TABLE 1 Reference Sample Example 1 Example 2 Example 1 No. (mg/dl)(mg/dl) (mg/dl)  1 56.3 60.7 55.1  2 38.5 46.2 37.3  3 53.3 59.4 54.7  447.5 45.0 45.8  5 48.8 54.6 47.2  6 44.7 47.6 42.1  7 62.4 70.5 63.7  874.2 79.5 66.9  9 44.9 53.7 41.0 10 53.1 56.6 52.2 Average Value 52.457.4 50.6 Coefficient of 0.969 0.931 — correlation to Reference Example1

From the result in the Table 1, it can be shown that the measurementvalue of cholesterol in HDL by the method of the present invention showshigh correlation with that obtained by the method of Reference Example 1(conventional method).

The measurement values obtained by the method of the present inventionare higher than those obtained In Reference Example 1. On the otherhand, it has been known that the value of cholesterol in HDL by themethod in Reference Example 1, phosphotungstic acid/magnesium saltprecipitation method, is rather lower than that obtained by the standardmethod, ultracentrifugal separation method. Therefore, the measuredvalues of cholesterol obtained by method of the present invention(particularly, by Example 1) are considered close to the measured valuesobtained by the standard method.

EXAMPLE 3

Amount of cholesterol contained in HDL in serum was measured by themethod of the present invention with the use of an automatic analyzerHitachi 7150 (Trade Name; manufactured and sold by Hitachi Ltd.).

[Samples]

10 Samples of fresh human serum.

[Reagents]

1st reagent (R-1): Antiserum to apolipoprotein B (12 mgAb/ml, 3% w/vmanufactured and sold by Boehringer Mannheim GmbH.) 4-Aminoantipyrine 1mM Tris-HCl buffer solution (pH 7.2) 100 mM 2nd reagent (R-2): COD 3Unit /ml CHE 3 Unit /ml POD 1 Unit /ml DAOS 1 mM Emalex NPL-30 0.05% w/vTris-HCl buffer solution (pH 7.2) 100 mM[Measuring Parameters (Measuring Conditions)]

Measurement method: 2 Point-end method [16]-[34]

Sample volume: 4 μl R-1 volume: 270 μl R-2 volume: 90 μl Measurementwave length: 700/600 nm Measurement temperature: 37° C.[Results]

The measurement result is shown in the table 2.

EXAMPLE 4

Amount of cholesterol contained in HDL in serum was measured by themethod of the present invention with the use of an automatic analyzerHitachi 7150 (Trade Name; manufactured and sold by Hitachi Ltd.).

[Samples]

Same as Example 3.

[Reagents]

1st reagent (R-1): Antiserum to apolipoprotein B (12 mgAb/ml, 3% w/vmanufactured and sold by Boehringer Mannheim GmbH.) 4-Aminoantipyrine 1mM Tris-HCl buffer solution (pH 7.2) 100 mM 2nd reagent (R-2): COD 3Unit /ml CHE 3 Unit /ml POD 1 Unit /ml DAOS 1 mM Triton X-405 0.1% w/vTris-HCl buffer solution (pH 7.2) 100 mM[Measuring Parameters (Measuring Conditions)]

Same as Example 3.

[Results]

The measurement result is also shown in the table 2.

Reference Example 2

On the serum samples used in Example 3, cholesterol contained in HDL wasmeasured by known phosphorus-tungstic acid/magnesium salt precipitationmethod with the use of HDL-Cholesterol E-Test Wako (Trade Name;manufactured and sold by Wako Pure Chemical Industries, Ltd.).

The measurement procedure was as the standard procedure explained in thebrochure of said kit.

[Results]

The measurement results is also shown in the table 2.

TABLE 2 Reference Sample Example 3 Example 4 Example 2 No. (mg/dl)(mg/dl) (mg/dl) 11 48.4 38.6 47.2 12 44.7 37.6 42.1 13 66.5 54.1 66.9 1456.6 45.5 52.6 15 43.5 40.2 41.8 16 36.1 37.6 34.2 17 56.3 45.1 54.2 1837.8 34.2 36.9 19 51.3 44.6 47.2 20 33.0 30.4 33.6 Average Value 47.440.8 45.7 Coefficient of 0.989 0.939 — correlation to Reference Example2

From the result in the Table 2, it can be shown that the measurementvalue of cholesterol in HDL by the method of the present invention showshigh correlation with that obtained by the method of Reference Example 2(conventional method).

As can be clearly understood from the disclosure in the specification,the present invention provides a method for measuring the objectiveconstituents contained in the specific lipoprotein, in a biologicalsample, capable to apply directly to an automatic analyzer withoutcarrying out any pretreatment operation for removing lipoproteins otherthan the specific lipoprotein. By applying the present invention, (i)the amount of one or more objective constituents contained in a specificlipoprotein can be measured by use of a conventional type of automaticanalyzer because the measurement can be carried out by using only 2kinds of reagent solutions, moreover, high accuracy measurement isconducted because the measured values are obtained with excellentreproducibility; (ii) the measurement in the present invention isconducted by 2-point end method, because, it is not required to conductthe step of re-dissolving the agglutinated lipoproteins other than thespecific lipoprotein to measure the optical absorbance of a uniformsolution for measuring the objective constituent contained in thespecific lipoprotein. Also, the present invention provides advantageouseffect to avoid any adverse effect to the measurement of opticalabsorbance caused by co-existed substances contained in the biologicalsample, thus the present invention contributes greatly in this field.

1. A kit for measurement, by two point-end assay by using anautoanalyzer, of cholesterol contained in the class of high densitylipoproteins in a biological sample containing at least one other classof lipoprotein selected from the group consisting of the chylomicronsclass, the class of very low density lipoproteins and the class of lowdensity lipoproteins, said kit for measurement comprising: a firstreagent composition comprising (i) a buffering agent, (ii) an antibodywhich does not bind with the class of high density lipoproteins butwhich binds with said at least one other class of lipoprotein containedin the biological sample to start an antibody bonding reaction and (iii)peroxidase, said antibody substantially preventing the cholesterol insaid at least one other class of lipoproteins from participating in areaction for measurement of cholesterol, wherein said first reagentcomposition does not contain cholesterol esterase and/or cholesteroloxidase; a second reagent composition separate from said first reagentcomposition, containing no antibody and comprising (i) a bufferingagent, (ii) cholesterol oxidase and (iii) cholesterol esterase; and4-aminoantipyrine and a reagent developing color by coupling with4-aminoantipyrine, wherein one of these is incorporated in the firstreagent composition and the other is incorporated in the second reagentcomposition.
 2. A kit for measurement, by two point-end assay by usingan autoanalyzer, of cholesterol contained in the class of high densitylipoproteins in a biological sample containing at least one other classof lipoprotein is selected from the group consisting of the chylomicronsclass, the class of very low density lipoproteins and the class of lowdensity lipoproteins, said kit for measurement comprises: a firstreagent composition comprising (i) a buffering agent, (ii) an antibodywhich does not bind with the class of high density lipoproteins butwhich binds with said at least one other class of lipoprotein containedin the biological sample to start an antibody binding reaction, (iii)4-aminoantipyrine and (iv) peroxidase, said antibody substantiallypreventing the cholesterol in said at least one other class oflipoproteins from participating in a reaction for measurement ofcholesterol, wherein said first reagent composition does not containcholesterol esterase and/or cholesterol oxidase; a second reagentcomposition separate from said first reagent composition, containing noantibody and comprising (i) a buffering agent, (ii) cholesterol oxidase,(iii) cholesterol esterase and (iv) a reagent developing color bycoupling with 4-aminoantipyrine.
 3. The kit as claimed in claim 1 or 2,wherein the antibody is at least one selected from the group consistingof anti-apolipoprotein A, anti-apolipoprotein B, anti-apolipoprotein C,anti-apolipoprotein E, anti-αlipoprotein antibody and anti-βlipoproteinantibody.
 4. The kit as claimed in claim 1 or 2, wherein theconcentration of the antibody is 0.001 to 10 mgAb/ml.